Research was conducted to investigate the biosynthesis of pertussis toxin (PT) as well as the structure and mechanism of action of the toxin. We initiated a study to address the question of whether vaccination can provide selective pressure for antigenic variation of PT and, if so, whether antigenic drift might result in decreased efficacy of acellular pertussis vaccines. We compared the sequence of the PT (ptx) genes of Japanese isolates of Bordetella pertussis obtained both before and after wide-spread use of acellular pertussis vaccines. The ptx regions from Tohama I (the vaccine strain), two clinical isolates obtained in 1979, and three clinical isolates obtained in the 1990's were sequenced. We found that all regions were identical indicating that antigenic drift had not occurred, at least in the strains analyzed. In addition, we examined whether antigenic variation of PT could affect the ability of vaccine to protect against B. pertussis. In order to do this, we cloned the ptx genes from B. bronchiseptica, and exchanged these genes for those found in B. pertussis. We then examined the ability of sera from mice immunized with PT originating from B. pertussis to neutralize the bronchiseptica toxin. We found that the bronchiseptica toxin was neutralized as efficiently as pertussis toxin, indicating that the multiple amino acid differences between bronchiseptica toxin and pertussis toxin, most of which occur on the exposed surface of the protein, did not affect the antigenicity of the protein. These results suggest if evolutionary drift of pertussis toxin does occur, it likely occurs very slowly and many of the alterations that would be allowed, since they would not affect biological function of the protein, do not significantly affect the antigenicity of the protein. We are continuing our work on the analysis of the secretion of PT from B. pertussis. Previously, we identified nine ptl genes that are essential for the secretion of the toxin. In order to gain further insight into the mechanism of secretion, we studied the ability of individual components of the toxin to be secreted. In order to do this, we constructed mutants of B. pertussis that expressed only the S1 subunit of the toxin or only the B oligomer of the toxin. We found that the B. oligomer was not secreted in the absence of the S1 subunit. In contrast, a considerable fraction of the total S1 subunit produced by the cell was found in the culture supernatant in the absence of the B oligomer, suggesting that the S1 subunit may be exported in the absence of the B oligomer. Studies are continuing to elucidate the sequence of events that occur during the secretion of pertussis toxin from B. pertussis.